Basic amino acid salts of phenoltetrabromophenolphthalein 3&#39;,3&#34;-disulfonic acid



Patented Nov. 10, 1970 3 539 586 BASIC AMINO ACID SAI .TS OF PHENOLTETRA- BROMOPHENOLPHTHALEIN 3',3"-DISULFONIC ACID Alexander Galat, Yonkers, N.Y. (1980 S. Ocean Drive, Hallendale, Fla. 33009) No Drawing. Filed July 18, 1968, Ser. No. 748,133 Int. Cl. C07d /38, 99/02 U.S. Cl. 260-309 5 Claims ABSTRACT OF THE DISCLOSURE Phenoltetrabromophenolphthalein 3', 3" disulfonic acid and basic amino acids such as lysine, ornithine, arginine and histidine, react to produce stable, non-hygroscopic salts characterized by essentially instantaneous solubility in water and extremely high water solubility.

Solutions of the resulting compounds are employed in accordance with known procedures to measure the liver function of human subjects and exhibit many advantages over materials hitherto employed in the performance of this diagnostic test.

A diagnostic test for liver function is of great clinical significance. The liver is involved in a large number of vital physiological functions such as detoxifications, fat metabolism, sugar storage, oxidations and many others and a test that measures the ability of the liver to perform these functions is, of course, extremely important.

At present the diagnostic reagent used to measure liver function is disodium phenoltetrabromophenolphthalein 3', 3"-disulfonate, also known as sulfobromophthalein sodium (this designation being commonly but not exclusively employed hereinafter), bromsulfophthalein sodium, hepartest, bromthalein, bromsulphalein sodium, brom-tetragnost, et cetera.

The test is performed as follows: A 5% aqueous solution of the dye is injected intravenousl into the subject at a preferred dosage of 5 mg. dye per kg. body weight. (The volume in ml. of a solution of this concentration to be injected is calculated by dividing the body weight of the subject in pounds by 22). The amount of dye remaining in the blood stream is measured after 45 minutes. If the liver function is normal the serum should be free of dye after this period of time.

Sulfobromophthalein sodium suifers from the great disadvantage of being only sparingly soluble in water. At room temperature its solubility is only about 5% and this is depressed quite sharply at lower temperatures and also is lowered in the presence of sodium ions commonly present in physiological fluids.

A number of serious drawbacks result from this low solubility. One of the most important is the fact that very large volumes of solution are required for injection. For example, a patient weighing 160470 pounds requires almost 8 m1. of solution. Such a large volume cannot be injected intramuscularly so the intravenous route must be used which is more dangerous and requires more skill.

Further, since the 5% solution of sulfobromophthalein sodium is at the limit of solubility of the compound, an

even slight decrease in the ambient temperature will result in formation of insoluble particles. Accordingly, ampoules of the reagent must be kept in a Warm place. If inadvertently or because of circumstances beyond control the temperature drops below 20 C. the ampoules must be carefully reheated until the solid redissolves, before an injection can be made. If due to oversight or carelessness the solution is injected without this precaution, severe reactions, sometimes fatal, may result.

As mentioned above, the reagent may also crystallize in the presence of other ions, particularly sodium, which tend to decrease the solubility of the dye.

Finally, in addition to being sparingly soluble, sulfobromophthalein sodium has the disadvantage of being slowly soluble. Thus, mechanical agitation and considerable time is required to dissolve this salt. As a result, the reagent cannot be put into ampoules in solid form to be dissolved just before use but must be supplied in solu tion form. This has several disadvantages. First, solutions of sulfobromophthalein sodium are ideal media for bacterial growth and must be sterilized with exceptional care. Second, since the volume of solution to be injected varies with the weight of the subject, it becomes necessary to supply the reagent in several volumes. Commercially, three sizes, 3, 7 and 10 mil. are supplied. Obviously, if the reagent were a highly and instantly soluble substance it could be supplied in solid form. This would be dissolved before use, thus eliminating the danger of bacterial contamination. Also, only one size ampoule would be needed since solid doses of different Weight could be dissolved in the same, small volume of water.

An object of this invention is to provide derivatives of sulfobromophthalein characterized by high aqueous solubility.

A further object of this invention is to provide derivatives of sulfobromophthalein characterized by rapid solubility in water.

Another object of this invention is to provide derivatives of sulfobromophthalein which, in aqueous solution, are unalfected by foreign ions.

An additional object of this invention is to provide new and improved diagnostic reagents for measuring liver function.

Yet a further object of this invention is to provide intramuscularly administrable diagnostic reagents for measuring liver function.

Other objects of this invention will become apparent as the description thereof proceeds.

The objects of my invention are accomplished by salts of sulfobromophthalein and a basic amino acid.

Basic amino acids comprise a well known and Well recognized class of organic compounds-see, for example, Fieser and Fieser, Organic Chemistry, Third Edition, p. 420 et seq. Basic amino acids are characterized by the presence of two basic groups per carboxyl group, such as two amino groups per carboxyl group exemplified by lysine (alpha, epsilon diamino caproic acid) and ornithine (alpha, delta diamino valeric acid) or by the presence of one amino group plus a second basic group of another type per carboxyl group exemplified by arginine (alpha amino delta guanidylvaleric acid) and histidine (alpha amino beta (Z-imidazoyl) propionic acid).

The formula of the compounds of my invention is shown below where lysine is chosen as the basic amino acid.

The high and essentially instantaneous aqueous solubility of the compounds of my invention is an entirely unexpected result.

It is well known, of course, that usually organic acids can be readily solubilized by the formation of various salts thereof (sodium, potassium, calcium, ammonium and amine salts, et cetera). However, when such salts of sulfobromophthalein are prepared only sparingly soluble compounds are formed. Thus, the low solubility of the sodium salt has already been discussed. The potassium, ammonium and calcium salts are even less soluble. Physiologically aceptable amines give salts of still lower solubility. For example, ethylenediamine, commonly used to solubilize acidic drugs, gives a sulfobromophthalein salt exhibiting an aqueous solubility of less than 1%.

Accordingly, conventionally used cations give very sparingly soluble salts with sulfobromophthalein and this explains the use of the sodium salt whose undesirably low solubility (5%) is still the highest that the prior art could produce.

I have discovered that salts of sulfobromophthalein with basic amino acids are stable, nonhygroscopic, instantly soluble compounds extremely soluble in water.

The preparation of these compounds can be illustrated by the following example:

A solution of 79.4 g. (0.1 mole) of phenoltetrabromophenolphthalein 3',3"-disulfonic acid (sulfobromophthalein) and 29.2 g. (0.2 mole) of l-lysinc in 100 ml. water was treated slowly with 400 ml. methanol. The crystalline precipitate, upon filtration, washing with methanol and drying weighed 67 g. The mother liquors were treated with further amounts of methanol until the total volume was one liter, which produced a second crop of the product. The combined yield was 94 g. (86% of thecry).

The new compound is instantly soluble in water. Its solubility is so high that it is difficult to measure it accurately. For example, it readily dissolves in its own weight of water, which makes it at least twenty times as soluble as the sodium salt. Of similarly high solubility are the ornithine, arginine and histidine salts.

Because of the high solubility thereof any amount of the compounds of this invention that may be practically required can be dissolved in only one ml. of water. For example, even a subject weighing 100 kg. (220 pounds) would require only 1 ml. of solution since the 500 mg.

of the reagent required dissolve readily in that volume of water. In contrast, the sodium salt would require 10 ml. of water to produce a solution containing the quantity of reagent required in testing the liver function of a subject of the weight.

The high solubility of the compounds of this invention results in much smaller volumes required for injection and accordingly makes possible the use of intramuscular injections. This is much more convenient and safer than intravenous injections.

Further, solutions produced using the compounds of this invention, even when very concentrated, do not crystallize at low temperatures. Thus, a 50% solution of the lysine salt when kept in a refrigerator for three days remained perfectly clear and suitable for injection without any reprocessing. In contrast, a 5% solution of the sodium salt similarly treated deposited a heavy crystalline precipitate which had to be redissolved by heating. As pointed out above, this property of the compounds of the present invention of remaining in solution regardless of variations in ambient temperature is very advantageous in that there is no possibility of solid material being inadvertently injected into the subject with the resulting possibility of severe side reactions.

Additionally, solutions of the compounds of the pres ent invention are not affected in any way by the presence of sodium or other ions. On the other hand, a solution of sulfobromophthalein sodium will crystallize in the presence of sodium ions due to the common ion effect, as well as in the presence of potassium and other ions.

Another advantage of the compounds of the present invention resides in their ability to dissolvealmost instantly upon contact with water. This property makes possible the dispensing of these compounds in solid form. When needed, the compound is dissolved to form a stock solution for many injections or ampouled in single doses for unit use. As mentioned above, this offers many advantages, such as freedom from bacterial contamination and the use of single volume units of the same size.

Of the various basic amino acids, I prefer to use l-ylsine in forming the compounds of this invention, this material being readily available at a reasonable cost. However, other basic amino acids can be used with equally satisfactory results. Among these may be mentioned orinthine, arginine and histidine.

I claim:

1. Basic amino acid salts of phenoltetrabromophenolphthalein 3',3"-disulfonic acid.

2. The alpha, epsilon diamino caproic acid salt of phenoltetrabromophenolphthalein 3,3"-disulfonic acid.

3. The alpha, delta diamino valeric acid salt of phenoltetrabromophenolphthalein 3,3"-disulfonic acid.

4. The alpha amino delta guanidylvaleric acid salt of phenoltetrabromophenolphthalein 3,3"-disulfonic acid.

5. The alpha amino beta (Z-imidizaloyl) propionic acid salt of phenoltetrabromophenolphthalein 3',3"-disulfonic acid.

References Cited Brauer et al.: Chem. Abs. 58-2730 (1963). Cohen et al.: Chem. Abs. 60-l1103 (1964).

ALEX MAZEL, Primary Examiner A. NARCAVAGE, Assistant Examiner US. Cl. X.R. 

